Project Summary/Abstract Ca2+ controls cardiac function by acting as the primary regulator of the sarcomeric contractile machinery and as a second messenger in the signal transduction pathways that control cardiac growth, metabolism and pathological remodeling. Ca2+ handling in striated muscle is tightly regulated by Ca2+ pumps in the sarcoplasmic reticulum (SR) and plasma membranes that maintain intracellular Ca2+ levels ~10,000-fold lower than extracellular and SR concentrations. Ca2+ release from the SR membrane transiently increases Ca2+ levels in the cytosol, triggering actomyosin cross-bridge formation within the sarcomere to generate contractile force. Reuptake of Ca2+ into the SR by sarcoplasmic reticulum Ca2+-ATPase (SERCA) is necessary for muscle relaxation and restores SR Ca2+ levels for subsequent contraction-relaxation cycles. SERCA thus serves as a central regulator of cardiac function, as well as the pathogenic signaling cascades that drive heart disease. The activity of SERCA in the heart is modulated by phospholamban (PLN), a tiny peptide that interacts with SERCA in the SR membrane and diminishes Ca2+ pump activity. We discovered that a cardiac-specific RNA annotated as a long noncoding RNA actually encodes a previously unrecognized micropeptide, which we named DWORF (Dwarf Open Reading Frame). During the initial funding period we showed that DWORF has a higher binding affinity for SERCA than PLN and that DWORF overexpression mitigates the contractile dysfunction associated with PLN overexpression, substantiating its role as a potent activator of SERCA. Additionally, using a mouse model of dilated cardiomyopathy, we showed that DWORF overexpression restores cardiac function and prevents the pathological remodeling and Ca2+ dysregulation. Our results established DWORF as a potent activator of SERCA within the heart and as an attractive candidate for a heart failure therapeutic. Recently, we discovered two PLN-related micropeptides, referred to as Endoregulin (ELN) and Another-regulin (ALN), which associate with specific SERCA isoforms, suggesting their involvement in SERCA-dependent Ca2+ signaling. Collectively, we refer to this family of inhibitory SERCA micropeptides as Regulins. Our discovery of the DWORF- Regulin micropeptides provides new inroads into our understanding of the mechanisms involved in cardiac contractility and function and points to unexplored roles of micropeptides in the control of cardiovascular physiology and pathology. Our hypothesis is that DWORF-Regulin micropeptides are critical for cellular homeostasis and stress adaptation in disease, such that these micropeptides can serve as therapeutic targets for cardiovascular and metabolic diseases. The overall goals of this proposal are to define the functions and regulatory protein-protein interactions of DWORF and Regulins in the cardiovascular system and evaluate their therapeutic significance.